CN103013821B - Avidin-biotin system cell patterning-based chip and preparation and applications thereof - Google Patents
Avidin-biotin system cell patterning-based chip and preparation and applications thereof Download PDFInfo
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- CN103013821B CN103013821B CN201210511033.3A CN201210511033A CN103013821B CN 103013821 B CN103013821 B CN 103013821B CN 201210511033 A CN201210511033 A CN 201210511033A CN 103013821 B CN103013821 B CN 103013821B
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Abstract
The invention discloses an avidin-biotin system cell patterning-based chip and preparation and applications thereof, belonging to the technical field of a biological micro system. The method comprises the steps: preparing a bioaffinity hexamethyl disilazane (HMDS) lattice array and bio-passivated polyethylene glycol pattering area by adopting photoetching and chemical precipitation methods; then forming a sandwich structure of biovine serum albumin-biotin, avidin and biotinylation cells on the bioaffinity HMDS lattice array, thereby directionally distributing cells and realizing the separation and locating of cells. The materials used in the method are simple, and the method is simple and convenient; the distribution of the cell arrays can be realized by controlling the size and distribution mode of the HMDS; the method is used for patterning various cells; single-cell arrays are not easily broken in the following detection and application of the chip; and the materials and biochemical substances have small influence on the activity of cells, thereby being used for detecting living cells.
Description
Technical field
The invention belongs to biological micro-systems technical field, be specifically related to a kind of based on the graphical chip of Avidin-Biotin system cells and preparation and application.
Background technology
Conventional medicament screening method determines by following the trail of the biochemical route of animal tissues's pathology the crucial biological substance that causes pathology, the target molecule that recycles this kind of material carries out drug screening, and the complexity that possesses skills, small throughput, experimentation on animals is many, drug dose is large, high in cost of production shortcoming.Micrographics technology not only provides a kind of alternative costliness, time-consuming zooperal research means for drug screening research, more research level has been brought up to even gene level of cell, and high-throughout feature shortens the cycle of drug screening and new drug development greatly.On cell levels, pattern technology can need to be constructed cell figure, fixed cell position according to detecting, thereby by real time, detect the reaction of cell to medicine quickly and accurately, carries out drug screening.
Utilize biochemical material to stimulate tissue regeneration, repair the important development direction that has become organizational project, the biochemical character at research interface plays vital effect to optimizing the performance of implantation site biochemical material.Determined the process such as attaching, growth, breeding of cell with form, rigidity and the molecular structure at the interface of cells contacting.Pattern technology provides efficient, a brand-new research platform for the biological process of research on interface.
Major disease diagnosis has quick, sensitive, high-throughout feature with disease somatotype requirement test set, due to the complicacy of biosystem, often needs at present tens biosensors to segment type by diagnosed disease.Pattern technology possesses multiple biological acceptor is integrated in to the potential ability in a slice addressable sensor, by the parallel detection to sample, simplifies medical diagnosis on disease flow process, for disease treatment tries to gain time precious to one.
Summary of the invention
The object of the invention is to utilize the strong keying action between avidin and vitamin H, a kind of separation of Avidin-Biotin system cells and localization method of utilizing is provided.
A kind of based on the graphical chip of Avidin-Biotin system cells, in substrate, be covered with the film that one deck has bioaffinity region and biological passive area simultaneously, described bioaffinity region is the hmds (Hexamethyldisilazane of cell yardstick, HMDS) lattice array, described biological passive area is polyoxyethylene glycol (polyethylene glycol, PEG) patterned area.
The above-mentioned preparation method based on the graphical chip of Avidin-Biotin system cells, comprises following steps:
(1) bioaffinity region preparation: spin coating photoresist material in substrate, photoresist material is carried out to photolithographic exposure, develop, then utilize chemical vapour deposition (Chemical Vapor Deposition, CVD), with sunk area after hmds filling photoresist developing, remove photoresist material, form hmds lattice array;
(2) biological passive area preparation: by standing in above-mentioned substrate immersion polyoxyethylene glycol solution of silane, make substrate exposed part cover biological passivating material polyoxyethylene glycol, obtain biological passive area.
Utilize above-mentioned method of carrying out cellular segregation and location based on the graphical chip of Avidin-Biotin system cells, it is characterized in that, comprise the following steps:
(1) preparation of cellular array: by cell surface biotin labeling, on chip, drip bovine serum albumin (Albumin from bovine serum, BSA)-vitamin H (biotin-BSA) solution, utilize the adhesivity of BSA, BSA is connected in HMDS lattice array, forms biotin-BSA lattice array;
(2) in biotin-BSA lattice array, evenly drip avidin, avidin is connected with vitamin H, form avidin lattice array;
(3) biotinylation cell is evenly seeded in avidin lattice array, hatch, obtain the sandwich structure of bovine serum albumin-vitamin H, avidin and biotinylation cell, the cell of non-specific adsorption on polyoxyethylene glycol region removed in cleaning, in hmds lattice array, obtain firmly cellular array, thereby oriented alignment cell, separation and the location of realizing cell.
Beneficial effect of the present invention is: 1) utilize the method for photoetching and CVD in substrate of glass, to carry out graphically, material therefor is simple, and method is easy, not high to requirement for experiment condition; 2) control of profit to the size of HMDS array and arrangement mode, can control arranging of (list) cellular array; 3) utilize NHS(N-N-Hydroxysuccinimide, N-Hydroxysuccinimide)-LC-Biotin carries out biotinylation to cell surface, can catch by graphical avidin, the method does not require the kind of cell, can use graphical with various cells; 4) utilize the strong keying action between avidin and vitamin H, cell is combined with substrate firmly, and in the subsequent detection application of chip, unicellular array is difficult for destroyed; 5) material therefor and biochemical substances are little on cytoactive impact, can make it for the detection of active somatic cell.
Accompanying drawing explanation
Fig. 1 is cellular array structure iron.
Fig. 2 is HMDS and PEG passive area graphic process figure.
Fig. 3 is unicellular preparation flow figure.
Embodiment
Below in conjunction with the drawings and specific embodiments, be described further.
Cellular array structure behind separated location, as shown in Figure 1.This invention is first carried out graphically at HMDS and PEG with photoetching and CVD in substrate of glass, then on HMDS array, utilizes the sandwich structure of Biotin-BSA, avidin and cell to form (list) cellular array.
The graphical treatment of 1.HMDS and PEG passive area:
With Piranha washing lotion ultrasonic cleaning slide glass, remove metal ion and the organism of slide surface; Spin coating photoresist material on the slide glass having cleaned, and front baking; Photoresist material is carried out to photolithographic exposure and carries out middle baking; In NaOH developing solution, develop and obtain photoetching offset plate figure post bake; Utilize chemical vapour deposition, cooling in HMDS steam, do not have photoresist material partly to deposit HMDS slide glass; Utilize acetone to remove photoresist, remove all photoresist materials, form HMDS lattice array; At PEG-Silane(silane) standing in solution, make slide glass exposed part cover biological passivating material PEG; Finally, on slide glass, obtain the hydrophobic HMDS lattice array good with bioadhesive, and at all the other local material PEG that is difficult to bioadhesion that covers, as shown in Figure 2.
2. the preparation of cellular array:
1) biotinylation of cell surface
Utilize NHS(N-N-Hydroxysuccinimide, N-Hydroxysuccinimide)-LC-Biotin carries out biotinylation to cell surface, NHS-Biotin is reacted with the amido of cell surface proteins, vitamin H is connected with cell surface proteins, make viable cell surface biological elementization.
2) assembling of cellular array
On the graphical slide glass of HMDS and PEG passive area, evenly drip the biotin-BSA solution of 20 μ g/ml, utilize the adhesivity of BSA, BSA is connected in HMDS lattice array, form biotin-BSA lattice array.In biotin-BSA lattice array, evenly drip the avidin solution of 20 μ g/ml, utilize the strong keying action between the vitamin H on avidin and BSA, avidin is connected with vitamin H, form avidin island array.By 5 * 10
6the biotinylated cell solution of/ml is evenly seeded on avidin island array,, hatch 30 minutes.Due to the strong keying action between avidin and vitamin H, cell is connected with avidin island array.The cell of non-specific adsorption on PEG region removed in cleaning, forms cellular array.Especially, when HMDS island array is on unicellular yardstick, the biotin-BSA forming on it, avidin lattice array all on unicellular yardstick, thereby after inoculating cell, form unicellular array, as shown in Figure 3.
Claims (2)
1. one kind based on the graphical chip of Avidin-Biotin system cells, it is characterized in that, in substrate, be covered with the film that one deck has bioaffinity region and biological passive area simultaneously, described bioaffinity region is the hmds lattice array of cell yardstick, and described biological passive area is polyoxyethylene glycol patterned area;
Describedly based on the graphical chip of Avidin-Biotin system cells, prepare according to the following steps:
(1) bioaffinity region preparation: spin coating photoresist material in substrate, photoresist material is carried out to photolithographic exposure, develop, then utilize chemical vapour deposition, with sunk area after hmds filling photoresist developing, remove photoresist material, form hmds lattice array;
(2) biological passive area preparation: by standing in above-mentioned substrate immersion polyoxyethylene glycol solution of silane, make substrate exposed part cover biological passivating material polyoxyethylene glycol, obtain biological passive area.
2. utilize the method for carrying out cellular segregation and location described in claim 1 based on the graphical chip of Avidin-Biotin system cells, it is characterized in that, comprise the following steps:
(1) preparation of cellular array: by cell surface biotin labeling, drip bovine serum albumin-biotin solution on chip, bovine serum albumin is connected in hmds lattice array, form bovine serum albumin-vitamin H lattice array;
(2) in bovine serum albumin-vitamin H lattice array, evenly drip avidin, form avidin lattice array;
(3) biotinylation cell is evenly seeded in avidin lattice array, biotinylation cell is captured by avidin, obtain the sandwich structure of bovine serum albumin-vitamin H, avidin and biotinylation cell, the cell of non-specific adsorption on polyoxyethylene glycol region removed in cleaning, in hmds lattice array, obtain firmly cellular array, thereby oriented alignment cell, separation and the location of realizing cell.
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| CN103013821B true CN103013821B (en) | 2014-11-26 |
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| CN1734265A (en) * | 2004-08-03 | 2006-02-15 | 中国科学院大连化学物理研究所 | A kind of cell inclusion analytical approach based on micro-fluidic chip |
| CN100575929C (en) * | 2007-05-08 | 2009-12-30 | 湖南大学 | Utilize one-dimensional microflow controlled biochip to detect the method for gene mutation in the cell |
| CN102053160B (en) * | 2009-11-06 | 2013-09-18 | 上海交通大学附属第一人民医院 | Micro-flow control cell chip capable of capturing various cells simultaneously |
| CN101955595B (en) * | 2010-08-11 | 2012-01-04 | 东南大学 | Method for guiding fixed-point cell growth by preparing chemical micro-patterns on surfaces of various materials |
| CN102174387A (en) * | 2011-01-25 | 2011-09-07 | 重庆大学 | Low-voltage direct-current controlled continuous flow cell electrofusion chip |
| CN102226168A (en) * | 2011-04-26 | 2011-10-26 | 武汉大学 | Method for capturing cells based on micro-nano fibers |
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